Use of proteins extractable from animal organs for the preparation of medicaments for the treatment of pathological conditions characterized by hyperproduction of tumor necrosis factor (TNF)

ABSTRACT

Proteins extractable with perchloric acid from mammal liver, in particular from goat liver, are capable of lowering blood levels of Tumor Necrosis Factor (TNF) and can be used for the treatment of multiple sclerosis, rheumatoid arthritis, septic shock and other pathologies characterized by TNF hyperproduction.

The present invention relates to the use of proteins extractable fromanimal organs for the preparation of medicaments for the treatment ofpathological conditions characterized by hyperproduction of TumorNecrosis Factor (TNF).

TNF, also known as cachectin, is a proinflammatory cytokine playing animportant role in starting, together with IL-1, the cascade of othercytokines and factors which trigger the immune response in infectionsand in cancer. This response is paramount for a complete resolution ofinfections and metastatic processes, but it can occur in an uncontrolledway, thus causing damage to the host. TNF hyperproduction is consideredto be involved in a number of pathological conditions, such as septicshock, tumor cachexia, autoimmune diseases (rheumatoid arthritis,multiple sclerosis), meningococcal septicemia, Crohn's disease, etc.

WO 92/10197 disclosed protein fractions extractable with perchloric acidfrom organs of mammals, and their use as anticancer agents. Within thesefractions, three main components could be identified, having molecularweights of 50, 14 and 10 KDa on gel electrophoresis. Hereinafter, thepurified extract containing these three components will be referred toas UK 101. The sequence of the 14 KDa component, which is the main, ifnot the only protein, responsible for the described activities, isreported in WO 96/02567 and it has turned out to be related to thatdescribed by other authors (Levy-Favatier, Eur. Biochem. 1903, 212 (3)665-73) who have assumed that the novel identified sequences belong tothe family of the proteins known as chaperonins, to which the HSPs (HeatShock Proteins) themselves belong.

The proteins described in WO 92/10197 and those of WO 96/02567(hereinafter referred to as UK 114) show properties not previouslyobserved in chaperonins or analogous proteins. Now it has been found, inparticular, that said proteins are capable of significantly lowering TNFblood levels and therefore they can be used for the treatment ofpathological conditions characterized by hyperproduction of TNF.

The invention relates particularly to the use of the purified protein UK114.

Moreover the invention comprises the use of proteins showing highhomology to UK 114, of at least 80%, especially of 90% or more.

The activity of the proteins UK 101 and UK 114 has been demonstrated invitro, on mononuclear leukocytes from peripheral blood and in vivo, byevaluating the effect of the administration of UK 101 on the productionby mouse splenocytes as reported hereinafter.

In Vitro Tests

Mononuclear leukocytes from peripheral blood (PBMC), at a concentrationof 1 million/ml, were stimulated in vitro with lipopolysaccharide (100/ng/ml), for 4 hour in the absence or in the presence of UK 114 (1 μg/mland 10 μg/ml).

TNF Levels Were Measured by ELISA

Results

TNF production by PBMC was inhibited by the addition of UK 114 in vitro.

The decrease was by 90% with a 1 μg/ml dose of UK 114 and by 70% with a10 μg/ml dose of UK 114.

In Vivo Tests

Treatment

Mice were treated with 100 μg/mouse of UK 101 on alternate days for 15days (7 injections).

TNF has been measured 48 hours after the first injection and 48 hoursafter the last administration.

Preparation of the Cells and TNF Measurements

Splenocytes (4×10⁶ cells/ml) were incubated in the presence of 10 μg/mlof the polyclonal mitogen Concanavalin-A (With-A), for 48 hours, at37°C., 5% CO₂.

The amount of produced TNF released into the supernatant has beenevaluated using an immunoenzymatic method (ELISA).

Results

Treatment with UK 101 significantly decreased TNF production by mousesplenocytes. The effect is evident 48 hours after the firstadministration and it is still present even 48 hours after the seventhadministration.

TNF, pg/ml physiological saline UK-101 48 hours after the 387 ± 72 247 ±30° 1st administration 48 hours after the 366 ± 46 264 ± 76,1° 7thadministration ° = p value

Therefore, UK 101 and UK 114 are capable of modifying the course of, orpreventing, pathological conditions characterized by TNFhyperproduction, such as multiple sclerosis, rheumatoid arthritis, tumorforms, septic shock, Crohn's disease, etc.

The proteins of the invention can be administered by means of suitableformulations, preferably injectable forms.

The procedure of administration (doses, frequency of administration,etc.) will be determined according to the circumstances, depending on anumber of factors such as the condition of the patient, stage of thedisease. Nevertheless a daily dosage ranging from 1 to 100 mg will besuitable.

TABLE (SEQ ID NO: 1) Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala1               5                   10 Lys Ala Pro Ala Ala Ile Gly ProTyr Ser Gln Ala         15                  20 Val Leu Val Asp Arg ThrIle Tyr Ile Ser Gly Gln 25                  30                  35 AspIle Asn Asp Phe Ser Ala Val Asn Asp Val Tyr Lys Gln Leu Gly Met Asp ProAla Ser Gly Gln Leu Val Pro             40                  45 Gly GlyVal Val Glu Glu Ala Lys Gln Ala Leu Thr    50                   55                   60 Asn Ile Gly Glu Ile LeuLys Ala Ala Gly Cys Asp                 65                  70 Phe ThrASn Val Val Lys Ala Thr Val Leu Leu Ala         75                     80 Asp Ile Asn Asp Phe Ser Ala Val AsnAsp Val Tyr 85                  90                  95 Lys Gln Tyr PheGln Ser Ser Phe Pro Ala Arg Ala             100                 105 AlaTyr Gln Val Ala Ala Leu Pro Lys Gly Gly Arg    110                  115                  120 Val Glu Ile Glu AlaIle Ala Val Gln Gly Pro Leu                   125                   130Thr Thr Ala Ser Val         135

TABLE (SEQ ID NO: 1) Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala1               5                   10 Lys Ala Pro Ala Ala Ile Gly ProTyr Ser Gln Ala         15                  20 Val Leu Val Asp Arg ThrIle Tyr Ile Ser Gly Gln 25                  30                  35 AspIle Asn Asp Phe Ser Ala Val Asn Asp Val Tyr Lys Gln Leu Gly Met Asp ProAla Ser Gly Gln Leu Val Pro             40                  45 Gly GlyVal Val Glu Glu Ala Lys Gln Ala Leu Thr    50                   55                   60 Asn Ile Gly Glu Ile LeuLys Ala Ala Gly Cys Asp                 65                  70 Phe ThrASn Val Val Lys Ala Thr Val Leu Leu Ala         75                     80 Asp Ile Asn Asp Phe Ser Ala Val AsnAsp Val Tyr 85                  90                  95 Lys Gln Tyr PheGln Ser Ser Phe Pro Ala Arg Ala             100                 105 AlaTyr Gln Val Ala Ala Leu Pro Lys Gly Gly Arg    110                  115                  120 Val Glu Ile Glu AlaIle Ala Val Gln Gly Pro Leu                   125                   130Thr Thr Ala Ser Val         135

What is claimed is:
 1. A method for treatment of pathologiescharacterized by TNF hyperproduction, comprising administering to ananimal in need of such treatment a treatment-effective amount of aprotein having the sequence of SEQ ID NO:1 or a protein having ahomology of at least 80% to a protein having the sequence of SEQ IDNO:1.
 2. The method according to claim 1, wherein the protein has thesequence of SEQ ID NO:1.
 3. A method for treatment of pathologiescharacterized by TNF hyperproduction, comprising administering to ananimal in need of such treatment a treatment-effective amount of aprotein extracted with perchloric acid from mammalian liver.